Acquiring Full-length CDNA Of Microsporum Canis Membrane Protein PQ-LRP Gene

Objective: Microsporosis canis due to microsporum canis is a contagious skin disease, mainly affecting human scalp and glabrous skin. Recently, accompanying with the popularity of house pets, its incidence rate is increasing. This group has successfully constructed the different gene library of microsporum canis by SSH preciously, filtered out 4 differently express genes sequenced the target genes and analyzed by real-time quantitative PCR .As a result, the membrane protein PQ-LRP gene was higher expressed in scalp-induced culture than glabrous skin. We suppose this high expression may be related to the frequent infection of children scalp with microsporum canis .At present, Scholars were keen on protease of microsporum canis, but there are few studies on the membrane protein. In this study, we try to get the full-length cDNA sequence of PQ-LRP by RACE, preliminary analysising of its structure and function, providing a theoretical basis for the future of its role in the pathogenesis of tinea capitis and new design target for drug development.Methods: To select strain of Microsporum canis tinea capitis (A518) as the experimental strain, extract total RNA. According to SMARTer RACE cDNA Amplification Kit User Manual instructions (Clontech, USA) . At first, to synthesize the first strand cDNA. According to the known PQ -LRP gene fragments, by PRIMER5.0 software design gene-specific primer (gene specific primer, GSP1) and GSP2 and nested primers NGSP1, NGSP2. GSP1 and GSP2 respectively completed the first round of PCR reaction with RACE primers UMP (the mixture of long and short fragments).The first round PCR products as template, respectively, using NGSP1, NGSP2 and nested primers NUP completed the second round PCR reaction. The second round PCR products were electrophoresed by agarose gel, and then the gel was purified and recovered. The purification were cloned into the pMD18-T vector, restriction enzyme digestion, positive clones were transformed into the E.coli DH5α. The bacterium with positive clones was sequence by Beijing Genomics Institute, and was spliced by the DNAStar software on the sequencing results. Finally, full-length cDNA of membrane protein PQ-LRP gene was obtained.Results:1.Microsporum canis tinea capitis strains A518, grew well and produced mycelium masses in the Sabouraud liquid medium.2.According to Microsporum canis PQ-LRP gene, designed 3'RACE primers and nested PCR primers, a length of 789bp sequence was amplified.3.According to Microsporum canis PQ-LRP gene, designed 5'RACE primers and nested PCR primers, a length of 181bp sequence was amplified.4.After spliced the overlaps between 5'and3'-RACE product, we obtained 1522bp sequence, which contain a complete open reading frame from 50bp to 1129bp, 1080bp of coding region, translates 359aa,and include 49bp of 5' untranslated region, and 393bp of 3' untranslated region. This sequence has been successfully submitted to GenBank (GenBank accession number JF767222).Conclusion:1.Obtained sequence is a full-length gene sequence, with a complete open reading frame;2.Similar comparative analysis showed that it shared 81% identity to Ma Trichophyton seven Tranmembrane protein 1 and Trichophyton tonsurans PQ-LRP in whole amino acid sequence and 79% identity to Trichophyton rubrum PQ-LRP in whole amino acid sequence.